Typically, peptides have been used in cell-based arrays to capture cells for study. Surfaces are printed with “RGD” peptides designed to bind integrins on a cell surface, but this system does not work with cells that don't have integrins such as non-adherent cells (e.g. leukocytes, lymphocytes) and does not allow controlled defined patterning with different cell types together in the same platform. The RGD system has the large disadvantage of initiating cell differentiation, thus changing the cell before it is analyzed, because integrins are the receptors that are involved in controlling differentiation and also activities that follow after integrin binding that relate to differentiation. (See Du, X. P. et al., Cell 1991, 65, 409-416; Xiong, J. P. et al., Science 2002, 296, 151-155.)
Indirect noncovalent attachment is demonstrated with a protein-protein attachment system in which DNA is indirectly attached to cells by a noncovalent linkage through an antibody-ligand interaction. (Bailey R C et al. J Am Chem Soc. 2007 February 21; 129(7):1959-67) An antibody specific for a target ligand on a cell is conjugated to DNA. The noncovalent linkage between the antibody and the ligand is based on hydrogen bonding, typical of protein-protein interactions. Single-stranded DNA (ssDNA) oligomers on antibodies specific for cell-surface ligands are attached to cells having those ligands, and the cells are in turn anchored to surfaces having ssDNA complementary oligomers that bind the partner strand on the cell to capture the cell.
Indirect covalent attachment of synthetic single-stranded DNA (ssDNA) strands to the surfaces of living cells was first shown using metabolic oligosaccharide engineering by Chandra, R. A. et al. Angew. Chem.-Int. Edit. 2006, 45, 896-901. The indirect covalency was through specific chemical handles (azides) that were introduced in cell surface sialic acids obtained after treating the cells with peracetylated N-azidoacetylmannosamine (Ac4ManNAz) (taking 3 days) prior to the introduction of the DNA. Phosphine-ssDNA conjugates were then covalently attached to the azide handle to form an amide bond (a Staudinger ligation reaction, E. Saxon, et al. Science 2000, 287) between the azido-sugar and the DNA. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar can be reacted with a biotinylated triarylphosphine to produce many stable cell-surface adducts. However, the covalent metabolic approach takes multiple days to prepare the cells for DNA attachment, and altering the cell surface sugars has metabolic effects on the cell that changes it before one gets a chance to analyze it. It is further limited to certain mammalian cells that possess sialic acid on their surface, and thus cannot be used for bacteria, plant cells, fungi, or many other animal cells.
Noncovalent attachment via antibodies and ligands at the cell surfaces will also activate the cells and thus perturb the cell before analysis can start, also tending to be weaker and “reversible” compared with a covalent attachment. In addition the antibody mechanism requires prevalence of ligand on the cell, and engineering an antibody specific for the ligand to affix sufficient DNA on the cell surface.
Noncovalent attachments via ligand interactions with an antibody at the cell-surface have been made where the antibody carries a strand of protein binding DNA. (Bailey R C et al. J Am Chem Soc. 2007 February 21; 129(7):1959-67). Both methods have the immediate disadvantage of activating the cell they seek to capture for study, thus transforming the thing of interest into something different before it can be analyzed. Overcoming the drawbacks in these early systems of DNA attachment on cell surfaces could transform this just described nacent field and offer valuable tools and manipulations previously not possible.